The present invention relates to conformationally constrained Nxcex1 backbone-cyclized somatostatin analogs cyclized via novel linkages, and to pharmaceutical compositions containing same.
Somatostatin Analogs
Somatostatin is a cyclic tetradecapeptide found both in the central nervous system and in peripheral tissues. It was originally isolated from mammalian hypothalamus and identified as an important inhibitor of growth hormone secretion from the anterior pituitary. Its multiple biological activities include inhibition of the secretion of glucagon and insulin from the pancreas, regulation of most gut hormones and regulation of the release of other neurotransmitters involved in motor activity and cognitive processes throughout the central nervous system (for review see Lamberts, Endocrine Rev., 1988, 9, 427). Additionally, somatostatin and its analogs are potentially useful antiproliferative agents for the treatment of various types of tumors.
Natural somatostatin (also known as Somatotropin Release Inhibiting Factor, SRIF) of the following structure:
H-Ala1-GlY2-Cys3-Lys4-Asn5-Phe6-Phe7-Trp8-Lys9-Thr10-Phe11-Thr12-Ser13-Cys14-OH
was first isolated by Guillemin and colleagues (Bruzeau et al. Science, 1973, 179, 78). It exerts its effect by interacting with a family of receptors. Recently, five receptor subtypes, termed SSTR1-5, have been identified and cloned. In its natural form, somatostatin has limited use as a therapeutic agent since it exhibits two undesirable properties: poor bioavailability and short duration of action. For this reason, great efforts have been made during the last two decades to find somatostatin analogs that will have superiority in either potency, biostability, duration of action or selectivity with regard to inhibition of the release of growth hormone, insulin or glucagon.
Structure-activity relation studies, spectroscopic techniques such as circular dichroism and nuclear magnetic resonance, and molecular modeling approaches reveal the following: the conformation of the cyclic part of natural somatostatin is most likely to be an antiparallel xcex2-sheet; Phe6 and Phe11 play an important role in stabilizing the pharmacophore conformation through hydrophobic interactions between the two aromatic rings; the four amino acids Phe7-Trp9-Lys9-Thr10 which are spread around the xcex2-turn in the antiparallel xcex2-sheet are essential for the pharmacophore; and (D)Trp8 is preferable to (L)Trp8 for the interactions with somatostatin receptor subtypes 2 through 5.
Nevertheless, a hexapeptide somatostatin analog containing these four amino acids anchored by a disulfide bridge: 
is almost inactive both in vitro and in vivo, although it has the advantage of the covalent disulfide bridge which replaces the Phe5-Phe11 hydrophobic interactions in natural somatostatin.
Four main approaches have been attempted in order to increase the activity of this hexapeptide somatostatin analog. (1) Replacing the disulfide bridge by a cyclization which encourages a cis-amide bond, or by performing a second cyclization to the molecule yielding a bicyclic analog. In both cases the resultant analog has a reduced number of conformational degrees of freedom. (2) Replacing the original residues in the sequence Phe7-(D)Trp8-Lys9-Thr10 with other natural or non-natural amino acids, such as replacing Phe7 with Tyr7 and Thr10 with Val10. (3) Incorporating additional functional groups from natural somatostatin with the intention that these new elements will contribute to the interaction with the receptor. (4) Eliminating one of the four amino acids Phe7-(D)Trp8-Lys9-Thr10 with the assumption that such analogs would be more selective.
The somatostatin analog, MK-678:
cyclo(N-Me-Ala6-Tyr7-(D)Trp8-Lys9-Val10-Phe)
is an example of a highly potent somatostatin analog designed using the first three approaches above (Veber, et al., Life Science, 1984, 34, 371). In this hexapeptide analog, a cis-amide bond is located between N-Me-Ala and Phe11, Tyr7 and Val10 replace Phe7 and Thr10 respectively, and Phe11 is incorporated from natural somatostatin.
Another group of somatostatin analogs (U.S. Pat. Nos. 4,310,518 and 4,235,886) includes Octreotide: 
the only approved somatostatin analog currently available. It was developed using the third approach described above. Here, (D)Phe5 and the reduced C-terminal Thr12-CH2OH are assumed to occupy some of the conformational space available to the natural Phe6 and Thr12, respectively.
The compound TT-232: 
is closely related to Octreotide and is an example of implementing the fourth approach described above. The lack of Thr10 is probably responsible for its high functional selectivity in terms of antitumor activity.
These examples of highly potent somatostatin analogs suggest that the phenylalanines in positions 6 and 11 not only play an important role in stabilizing the pharmacophore conformation but also have a functional role in the interaction with the receptor. It is still an open question whether one phenylalanine (either Phe6 or Phe11) is sufficient for the interaction with the receptor or whether both are needed.
It is now known that the somatostatin receptors constitute a family of five different receptor subtypes (Bell and Reisine, Trends Neurosci., 1993, 16, 34-38), which may be distinguished on the basis of their tissue specificity and/or biological activity. Somatostatin analogs known in the art may not provide sufficient selectivity or receptor subtype selectivity, particularly as anti-neoplastic agents (Reubi and Laissue, TIPS, 1995, 16, 110-115).
Symptoms associated with metastatic carcinoid tumors (flushing and diarrhea) and vasoactive intestinal peptide (VIP) secreting adenomas (watery diarrhea) are treated with somatostatin analogs. Somatostatin has been also approved for the treatment of severe gastrointestinal hemorrhages. Somatostatin may also be useful in the palliative treatment of other hormone-secreting tumors (e.g., pancreatic islet-cell tumors and acromegaly) and hormone dependent tumors (e.g., chondrosarcoma and osteosarcoma) due to its antisecretory activity.
Improved Peptide Analogs
As a result of major advances in organic chemistry and in molecular biology, many bioactive peptides can now be prepared in quantities sufficient for pharmacological and clinical utilities. Thus in the last few years new methods have been established for the treatment and therapy of illnesses in which peptides have been implicated. However, the use of peptides as drugs is limited by the following factors:. a) their low metabolic stability towards proteolysis in the gastrointestinal tract and in serum; b) their poor absorption after oral ingestion, in particular due to their relatively high molecular mass or the lack of specific transport systems or both; c) their rapid excretion through the liver and kidneys; and d) their undesired side effects in non-target organ systems, since peptide receptors can be widely distributed in an organism.
It would be most beneficial to produce conformationally constrained peptide analogs overcoming the drawbacks of the native peptide molecules, thereby providing improved therapeutic properties.
A novel conceptual approach to the conformational constraint of peptides was introduced by Gilon, et al., (Biopolymers, 1991, 31, 745) who proposed backbone to backbone cyclization of peptides. The theoretical advantages of this strategy include the ability to effect cyclization via the carbons or nitrogens of the peptide backbone without interfering with side chains that may be crucial for interaction with the specific receptor of a given peptide. While the concept was envisaged as being applicable to any linear peptide of interest, in point of fact the limiting factor in the proposed scheme was the availability of suitable building units that must be used to replace the amino acids that are to be linked via bridging groups. The actual reduction to practice of this concept of backbone cyclization was prevented by the inability to devise any practical method of preparing building units of amino acids other than glycine (Gilon et al., J. Org. Chem., 1992, 587, 5687).
Further disclosures by Gilon and coworkers (WO 95/33765 and WO 97/09344) provided methods for producing building units required in the synthesis of backbone cyclized peptide analogs. Recently, the successful use of these methods to produce backbone cyclized peptide analogs having somatostatin activity was also disclosed (WO 98/04583). All of these methods are incorporated herein in their entirety, by reference.
According to the present invention, novel peptide analogs, which are characterized in that they incorporate novel building units with bridging groups attached to the alpha nitrogens of alpha amino acids, have now been generated. Specifically, these compounds are backbone cyclized somatostatin analogs comprising a peptide sequence of four to twelve amino acids that incorporates at least one building unit, said building unit containing one nitrogen atom of the peptide backbone connected to a bridging group comprising an amide, thioether, thioester or disulfide, wherein the at least one building unit is connected via said bridging group to form a cyclic structure with a moiety selected from the group consisting of a second building unit, the side chain of an amino acid residue of the sequence or the N-terminal amino acid residue. Preferably, the peptide sequence incorporates five to eight amino acids.
Heretofore conformationally constrained backbone cyclized somatostatin analogs had selectivity predominantly to receptor subtype 5. These analogs were of limited therapeutic or diagnostic utility.
According to the present invention it is now disclosed that more preferred analogs are hexapeptide analogs with improved selectivity to the SST subtype 3 rather than subtype 5. Most preferred analogs include novel octapeptide analogs of somatostatin which display receptor selectivity to SST subtypes 2 and 5. Additional more preferred somatostatin analogs may advantageously include bicyclic structures containing at least one cyclic structure connecting two building units and a second cyclic structure which is selected from the group consisting of side-chain to side-chain; backbone to backbone and backbone to end. Some of these bicyclic analogs display receptor selectivity to the SST subtype 2.
For certain hexapeptide preferred analogs of the present invention (denoted herein PTR numbers 3123, 3113 and 3171), the amino acid Asn was substituted by the backbone Phe building unit at position 5. The configuration substitution of the native L-Trp at position 8 to D-Trp was made to improve the stability of the analog. The Thr residue at position 10 was substituted by the corresponding backbone Phe building unit. The unique configuration substitution at position 9 from L-Lys to D-Lys as shown in PTRs 3123 and 3171 in comparison to PTR 3113 imparts improved selectivity of binding to the SST receptor subtype SSTR3 rather than SSTR5.
The most preferred backbone cyclized somatostatin analogs of the invention are described in table 1:
where X is xe2x80x94NH2 or xe2x80x94OH and the bridging group extends between the two building units or as indicated below: For PTR 3171 and PTR 3173, the asterisk denotes that the bridging group is connected between the Nxcex1-xcfx89-functionalized derivative of an amino acid and the N terminus of the peptide. For PTR 3197 and PTR 3207, the asterisk denotes that the bridging group is connected between the Nxcex1-xcfx89-functionalized derivative of an amino acid and the side chain of the Cys residue. PTR 3205 is a bicyclic compound in which one bridge connects the two building units (Phe-C3 and Phe-N3) and the second is a disulfide bridge formed between the two Cys residues. SSTR indicates the somatostatin receptor subtypes to which each analog is selective.
These backbone cyclized somatostatin peptide analogs are prepared by incorporating at least one Nxcex1-xcfx89-functionalized derivative of an amino acids into a peptide sequence and subsequently selectively cyclizing the functional group with one of the side chains of the amino acids in the peptide sequence or with another xcfx89-functionalized amino acid derivative. The Nxcex1-xcfx89-functionalized derivative of amino acids preferably have the following formula: 
wherein X is a spacer group selected from the group consisting of alkylene, substituted alkylene, arylene, cycloalkylene and substituted cycloalkylene; Rxe2x80x2 is an amino acid side chain, optionally bound with a specific protecting group; B is a protecting group selected from the group consisting of alkyloxy, substituted alkyloxy, or aryl carbonyls; and G is a functional group selected from the group consisting of amines, thiols, alcohols, carboxylic acids and esters, aldehydes, alcohols and alkyl halides; and A is a specific protecting group of G.
Preferred building units are the co-functionalized amino acid derivatives wherein X is alkylene; G is a thiol group, an amine group or a carboxyl group; Rxe2x80x2 is phenyl, methyl or isobutyl; with the proviso that when G is an amine group, Rxe2x80x2 is other than H. Further preferred are xcfx89-functionalized amino acid derivatives wherein Rxe2x80x2 is protected with a specific protecting group.
More preferred are xcfx89-functionalized amino acid derivatives wherein G is an amino group, a carboxyl group, or a thiol group of the following formulae: 
wherein X, Rxe2x80x2 and B are as defined above.
The most striking advantages of these methods are:
1) cyclization of the peptide sequence is achieved without compromising any of the side chains of the peptide thereby decreasing the chances of sacrificing functional groups essential for biological recognition and function.
2) optimization of the peptide conformation is achieved by allowing permutation of the bridge length, direction, and bond type (e.g., amide, disulfide, thioether, thioester, etc.) and position of the bond in the ring.
3) when applied to cyclization of linear peptides of known activity, the bridge can be designed in such a way as to minimize interaction with the active region of the peptide and its cognate receptor. This decreases the chances of the cyclization arm interfering with recognition and function, and also creates a site suitable for attachment of tags such as radioactive tracers, cytotoxic drugs, light capturing substances, or any other desired label.
Backbone cyclized analogs of the present invention may be used as pharmaceutical compositions and in methods for the treatment of disorders including: cancers, endocrine disorders, gastrointestinal disorders, pancreatitis, post-surgical pain, and all types of inflammation. The pharmaceutical compositions comprising pharmacologically active backbone cyclized somatostatin agonists or antagonists and a pharmaceutically acceptable carrier or diluent represent another embodiment of the invention, as do the methods for the treatment of cancer or endocrine disorders and gastrointestinal disorders and inflammation using such compositions. The pharmaceutical compositions according to the present invention advantageously comprise at least one backbone cyclized peptide analog which is selective for one or two somatostatin receptor subtypes. These pharmaceutical compositions may be administered by any suitable route of administration, including topically or systemically. Preferred modes of administration include but are not limited to parenteral routes such as intravenous and intramuscular injections, as well as via nasal or oral ingestion.
Backbone cyclized analogs of the present invention may also be used as pharmaceutical compositions in methods for diagnosing cancer and imaging the existence of tumors or their metastases. The methods for diagnosis of cancer comprise administering to a patient a backbone cyclic analog or analogs labeled with a detectable probe which is selected from the group consisting of a radioactive isotope and a non-radioactive tracer. The methods for the diagnosis or imaging of cancer using such compositions represent another embodiment of the invention.
The compounds herein described may have asymmetric centers. All chiral, diastereomeric, and racemic forms are included in the present invention. Many geometric isomers of olefins and the like can also be present in the compounds described herein, and all such stable isomers are contemplated in the present invention.
By xe2x80x9cstable compoundxe2x80x9d or xe2x80x9cstable structurexe2x80x9d is meant herein a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent.
As used herein and in the claims, xe2x80x9calkylxe2x80x9d or xe2x80x9calkylenylxe2x80x9d is intended to include both branched and straight-chain all saturated aliphatic hydrocarbon groups having one to ten carbon atoms; xe2x80x9calkenylxe2x80x9d is intended to include hydrocarbon chains of either a straight or branched configuration having two to ten carbon atoms and one or more unsaturated carbon-carbon bonds which may occur in any stable point along the chain, such as ethenyl, propenyl, and the like; and xe2x80x9calkynylxe2x80x9d is intended to include hydrocarbon chains of either a straight or branched configuration having from two to ten carbon atoms and one or more triple carbon-carbon bonds which may occur in any stable point along the chain, such as ethynyl, propynyl, and the like.
As used herein and in the claims, xe2x80x9carylxe2x80x9d is intended to mean any stable 5- to 7-membered monocyclic or bicyclic or 7- to 14-membered bicyclic or tricyclic carbon ring, any of which may be saturated, partially unsaturated or aromatic, for example, phenyl, naphthyl, indanyl, or tetrahydronaphthyl tetralin, etc.
As used herein and in the claims, xe2x80x9calkyl halidexe2x80x9d is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having the one to ten carbon atoms, wherein 1 to 3 hydrogen atoms have been replaced by a halogen atom such as Cl, F, Br, and I.
As used herein and in the claims, the phrase xe2x80x9ctherapeutically effective amountxe2x80x9d means that amount of novel backbone cyclized peptide analog or composition comprising same to administer to a host to achieve the desired results for the indications described herein, such as but not limited of inflammation, cancer, endocrine disorders and gastrointestinal disorders.
The term, xe2x80x9csubstitutedxe2x80x9d as used herein and in the claims, means that any one or more hydrogen atoms on the designated atom is replaced with a selection from the indicated group, provided that the designated atom""s normal valency is not exceeded, and that the substitution results in a stable compound.
When any variable (for example R, X, Z, etc.) occurs more than one time in any constituent or in any Formula herein, its definition on each occurrence is independent of its definition at every other occurrence. Also, combinations of substituents and/or variables are permissible only if such combinations result in stable compounds.
As used herein xe2x80x9cpeptidexe2x80x9d indicates a sequence of amino acids linked by peptide bonds. The somatostatin peptide analogs of this invention comprise a sequence of amino acids of 4 to 24 amino acid residues, preferably 6 to 14 residues, each residue being characterized by having an amino and a carboxy terminus.
A xe2x80x9cbuilding unitxe2x80x9d indicates an Nxcex1 derivatized xcex1 amino acid of the general Formula No. 5: 
wherein X is a spacer group selected from the group consisting of alkylene, substituted alkylene, arylene, cycloalkylene and substituted cycloalkylene; Rxe2x80x2 is an amino acid side chain, optionally bound with a specific protecting group; and G is a functional group selected from the group consisting of amines, thiols, alcohols, carboxylic acids and esters, and alkyl halides; which is incorporated into the peptide sequence and subsequently selectively cyclized via the functional group G with one of the side chains of the amino acids in said peptide sequence or with another xcfx89-functionalized amino acid derivative.
The methodology for producing the building units is described in international patent applications PCT/IB95/00455 and PCT/IL97/00261, both of which are expressly incorporated herein by reference thereto for further details of this methodology. The building units are abbreviated by the three letter code of the corresponding modified amino acid followed by the type of reactive group (N for amine, C for carboxyl), and an indication of the number of spacing methylene groups. For example, Gly-C2 describes a modified Gly residue with a carboxyl reactive group and a two carbon methylene spacer, and Phe-N3 designates a modified phenylalanine group with an amino reactive group and a three carbon methylene spacer.
As used herein xe2x80x9cbackbone cyclic peptidexe2x80x9d denotes an analog of a linear peptide which contains at least one building unit that has been liked to form a bridge via the alpha nitrogen of the peptide backbone to another building unit, or to another amino acid in the sequence.
Certain abbreviations are used herein to describe this invention and the manner of making and using it. For instance, AcOH refers to acetic acid, Ada refers to adamantanacetyl, Adac refers to adamantanecarbonyl, Alloc refer to allyloxycarbonyl, Boc refers to the t-butyloxycarbonyl radical, BOP refers to benzotriazol-1-yloxy-tris-(dimethylamino)phosphonium hexafluorophosphate, BSA refers to bovine serum albumin, Cbz refers to the carbobenzyloxy radical, DCC refers to dicyclohexylcarbodiimide, DCM refers to dichloromethane, Dde refers to 1-(4,4-dimethy12,6-dioxocyclohex-1-ylidene-ethyl), DIEA refers to diisopropyl-ethyl amine, DMF refers to dimethyl formamide, DPPA refers to diphenylphosphoryl azide, Dtc refers to 5,5-dimethylthiazolidine-4-carboxylic acid, EDC refers to N-ethyl-Nxe2x80x2-(dimethylaminopropyl)-carbodiimide, EDT refers to ethanedithiol, Fmoc refers to the fluorenylmethoxycarbonyl radical, HATU refers to [0-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate, HBTU refers to 1-hydroxybenztriazolyltetramethyl-uronium hexafluorophosphate, HF refers to hydrofluoric acid, HOBT refers to 1-hydroxybenzotriazole, HPLC refers to high pressure liquid chromatography, MALDI-TOF MS refers to matrix-assisted laser desorption, time-of-flight mass spectrometry, Mts refers to the 4-methoxy-2,3,6-trimethylbenzenzsulfonyl, NMM refers to N-methylmorpholine, NMP refers to 1-methyl-2-pyrolidonone, PyBOP refers to Benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate, PyBrOP refers to Bromo-tris-pyrrolidino-phosphonium hexafluorophosphate, RT refers to room temperature, SRIF refers to Somatotropin Release Inhibitory Factor, TBTU refers to 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate, t-Bu refers to the tertiary butyl radical, TFA refers to trifluoroacetic acid.
The amino acids used in this invention are those which are available commercially or are available by routine synthetic methods. Certain residues may require special methods for incorporation into the peptide, and either sequential, divergent and convergent synthetic approaches to the peptide sequence are useful in this invention. Natural coded amino acids and their derivatives are represented by three-letter codes according to IUPAC conventions. When there is no indication, the L isomer was used. The D isomers are indicated by xe2x80x9cDxe2x80x9d before the residue abbreviation. List of Non-coded amino acids: Abu refers to 2-aminobutyric acid, Aib refers to 2-amino-isobutyric acid, b-Ala refers to b-Alanine, ChxGly refers to cyclohexyl Glycine, GABA refers to gama amino butyric acid, Hcys refer to homocystein, (p-Cl)Phe refers to para chloro Phenylalanine, (p-NH2)Phe refers to para amino Phenylalanine, (p-F)Phe refers to para fluoro Phenylalanine, (p-NO2)Phe refers to para nitro Phenylalanine, 1Nal refers to 1-naphthylalanine, 2Nal refers to 2-naphthylalanine, Nva refers to norvaline, Thi refers to thienylalanine.
According to the present invention peptide analogs are cyclized via bridging groups attached to the alpha nitrogens of amino acids that permit novel non-peptidic linkages. In general, the procedures utilized to construct such peptide analogs from their building units rely on the known principles of peptide synthesis; most conveniently, the procedures can be performed according to the known principles of solid phase peptide synthesis. The innovation requires replacement of one or more of the amino acids in a peptide sequence by novel building units of the general Formula: 
wherein R is the side chain of an amino acid, X is a spacer group and G is the functional end group by means of which cyclization will be effected. The side chain R is the side chain of any natural or synthetic amino acid that is selected to be incorporated into the peptide sequence of choice. X is a spacer group that is selected to provide a greater or lesser degree of flexibility in order to achieve the appropriate conformational constraints of the peptide analog. Such spacer groups include alkylene chains, substituted, branched and unsaturated alkylenes, arylenes, cycloalkylenes, and unsaturated and substituted cycloalkylenes. Furthermore, X and R can be combined to form a heterocyclic structure.
The terminal (xcfx89) functional groups to be used for cyclization of the peptide analog include but are not limited to:
a. Amines, for reaction with electrophiles such as activated carboxyl groups, aldehydes and ketones (with or without subsequent reduction), and alkyl or substituted alkyl halides.
b. Alcohols, for reaction with electrophiles such as activated carboxyl groups.
c. Thiols, for the formation of disulfide bonds and reaction with electrophiles such as activated carboxyl groups, and alkyl or substituted alkyl halides.
d. 1,2 and 1,3 Diols, for the formation of acetals and ketals.
e. Alkynes or Substituted Alkynes, for reaction with nucleophiles such as amines, thiols or carbanions; free radicals; electrophiles such as aldehydes and ketones, and alkyl or substituted alkyl halides; or organometallic complexes.
f. Carboxylic Acids and Esters, for reaction with nucleophiles (with or without prior activation), such as amines, alcohols, and thiols.
g. Alkyl or Substituted Alkyl Halides or Esters, for reaction with nucleophiles such as amines, alcohols, thiols, and carbanions (from active methylene groups such as acetoacetates or malonates); and formation of free radicals for subsequent reaction with alkenes or substituted alkenes, and alkynes or substituted alkynes.
h. Alkyl or Aryl Aldehydes and Ketones for reaction with nucleophiles such as amines (with or without subsequent reduction), carbanions (from active methylene groups such as acetoacetates or malonates), diols (for the formation of acetals and ketals).
i. Alkenes or Substituted Alkenes, for reaction with nucleophiles such as amines, thiols, carbanions, free radicals, or organometallic complexes.
j. Active Methylene Groups, such as malonate esters, acetoacetate esters, and others for reaction with electrophiles such as aldehydes and ketones, alkyl or substituted alkyl halides.
It will be appreciated that during synthesis of the peptide these reactive end groups, as well as any reactive side chains, must be protected by suitable protecting groups.
Suitable protecting groups for amines are alkyloxy, substituted alkyloxy, and aryloxy carbonyls including, but not limited to, tert butyloxycarbonyl (Boc), Fluorenylmethyloxycarbonyl (Fmoc), Allyloxycarbonyl (Alloc) and Benzyloxycarbonyl (Z).
Carboxylic end groups for cyclizations may be protected as their alkyl or substituted alkyl esters or thio esters or aryl or substituted aryl esters or thio esters. Examples include but are not limited to tertiary butyl ester, allyl ester, benzyl ester, 2-(trimethylsilyl)ethyl ester and 9-methyl fluorenyl.
Thiol groups for cyclizations may be protected as their alkyl or substituted alkyl thio ethers or disulfides or aryl or substituted aryl thio ethers or disulfides. Examples of such groups include but are not limited to tertiary butyl, trityl(triphenylmethyl), benzyl, 2-(trimethylsilyl)ethyl, pixyl(9-phenylxanthen-9-yl), acetamidomethyl, carboxymethyl, 2-thio-4-nitropyridyl.
It will further be appreciated by the artisan that the various reactive moieties will be protected by different protecting groups to allow their selective removal. Thus, a particular amino acid will be coupled to its neighbor in the peptide sequence when the NI is protected by, for instance, protecting group A. If an amine is to be used as an end group for cyclization in the reaction scheme the Nxcfx89 will be protected by protecting group B, or an xcex5 amino group of any lysine in the sequence will be protected by protecting group C, and so on.
The coupling of the amino acids to one another is performed as a series of reactions as is known in the art of peptide synthesis. Novel building units of the invention, namely the Nxcex1-xcfx89-functionalized amino acid derivatives are incorporated into the peptide sequence to replace one or more of the amino acids. If only one such Nxcex1-xcfx89-functionalized amino acid derivative is selected, it will be cyclized to a side chain of another amino acid in the sequence or to either of the two terminal amino acids of the peptide sequence. For instance: (a) an Nxcex1-(xcfx89-amino alkylene) amino acid can be linked to the carboxyl group of an aspartic or glutamic acid residue; (b) an Nxcex1-(xcfx89-carboxylic alkylene) amino acid can be linked to the xcex5-amino group of a lysine residue; (c) an Nxcex1-(xcfx89-thio alkylene) amino acid can be linked to the thiol group of a cysteine residue; and so on. A more preferred embodiment of the invention incorporates two such Nxcex1-xcfx89-functionalized amino acid derivatives which may be linked to one another to form N-backbone to N-backbone cyclic peptide analogs. Three or more such building units can be incorporated into a peptide sequence to create bicyclic peptide analogs as will be elaborated below. Thus, peptide analogs can be constructed with two or more cyclizations, including N-backbone to N-backbone, as well as backbone to side-chain or any other peptide cyclization.
As stated above, the procedures utilized to construct somatostatin analogs of the present invention from novel building units generally rely on the known principles of peptide synthesis. However, it will be appreciated that accommodation of the procedures to the bulkier building units of the present invention may be required. Coupling of the amino acids in solid phase peptide chemistry can be achieved by means of a coupling agent such as but not limited to dicyclohexycarbodiimide (DCC), bis(2-oxo-3-oxazolidinyl) phosphinic chloride (BOP-Cl), benzotriazolyl-N-oxytrisdimethyl-aminophosphonium hexafluoro phosphate (BOP), 1-oxo-1-chlorophospholane (Cpt-Cl), hydroxybenzotriazole (HOBT), or mixtures thereof.
It has now been found that coupling of the subsequent amino acid to the bulky building units of the present invention may require the use of additional coupling reagents including, but not limited to: coupling reagents such as PyBOP (Benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate), PyBrOP (Bromo-tris-pyrrolidino-phosphonium hexafluorophosphate), HBTU (2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluoro-phosphate), TBTU (2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate).
Novel coupling chemistries may be used, such as preformed urethane-protected N-carboxy anhydrides (UNCA""S), preformed acyl halides most preferably acyl chlorides.
Advantageously, it is also possible to use in situ generated amino acid chlorides. Such coupling may take place at room temperature and also at elevated temperatures, in solvents such as toluene, DCM (dichloromethane), DMF (dimethylformamide), DMA (dimethylacetamide), NMP (N-methyl pyrrolidinone), dioxane, tetrahydrofuran, diglyme and 1,3 dichloropropane, or mixtures of the above.
The preferred backbone cyclized somatostatin analogs of the present invention are now described.
One embodiment has the following formula: 
wherein m and n are 1 to 5;
X designates a terminal carboxy acid, amide or alcohol group;
R5 is GABA, Gly, b-Ala, 5-amino pentanoic acid or amino hexanoic acid;
R6 is (D)- or (L)-Phe or Tyr;
R7 is (D)- or (L)-Trp,(D)- or (L)-Phe, (D)- or (L)-1Nal or (D)- or (L)-2Nal, or Tyr;
R8 is (D)- or (L)-Trp;
R9 is (D)- or (L)-Lys;
R10 is Thr, Gly, Abu, Ser, Cys, Val, (D)- or (L)-Ala, or (D)- or (L)-Phe;
R11 is (D)- or (L)-Phe,(D)- or (L)-Ala, or Cys;
R12 is Gly, Val, Leu, (D)- or (L)-Phe or 1Nal or 2Nal; and
Y2 is amide, thioether or thioester.
A most preferred compound according to this embodiment is denoted PTR 3173 wherein the residues are as follows:
R5 is GABA;
R6 is Phe;
R7 is Trp;
R8 is (D)Trp;
R9 is Lys;
R10 is Thr;
R11 is Phe;
R12 is Gly; and
Y2 is amide.
Another embodiment has the general formula: 
wherein: m and n are 1 to 5
X designates a terminal carboxy acid, amide or alcohol group;
R6 is(D)- or (L)-Phe, or (D)- or (L)-Ala;
R7 is Tyr, (D)- or (L)-Ala, or (D)- or (L)-Phe;
R10 is Thr, Val, Ser, or Cys;
R11 is Val, (D)- or (L)-1Nal, (D)- or (L)-2Nal , or (D) or (L)-Phe;
R12 is Gly, (D)- or (L)-Ala, or (D) or (L)-Phe; and
Y2 is amide, thioether, thioester or disulfide.
Preferably:
R6 is(D)- or (L)-Phe;
R7 is Tyr or Phe;
R10 is Thr, Val or Ser;
R11 is Val, 1Nal or 2Nal;
R12 is Gly; and
Y2 is amide.
Yet another embodiment has the general formula: 
wherein: m and n are 1 to 5
X designates a terminal carboxy acid, amide or alcohol group;
R6 is (D)- or (L)-Phe, or (D)- or (L)-Ala;
R7 is Tyr or (D)- or (L)- Phe;
R8 is (D)- or (L)- Trp, (D)- or (L)-1Nal or (D)- or (L)-2Nal;
R10 is Thr, Val, Ser, or Cys;
R11 is Gly or (D) or (L)-Phe;
R12 is Thr, GABA, (D)- or (L)-1Nal, (D)- or (L)-2Nal, or (D) or (L)-Phe; and
Y2 is amide, thioether, thioester or disulfide.
Preferably,
R6 is(D)- or (L)-Phe;
R7 is Tyr;
R8 is (D)Trp, (D)1Nal or (D)2Nal;
R10 is Val;
R11 is Gly;
R12 is Thr, 1Nal or 2Nal; and
Y2 is amide.
One more preferred embodiment has the following formula: 
wherein m and n are 1 to 5;
X designates a terminal carboxy acid, amide or alcohol group;
R4 is absent or is a terminal group of one to four amino acids;
R5 is 1Nal, 2Nal, xcex4-Asp (Ind), Gly, Tyr, (D)- or (L)-Ala, or (D)- or (L)-Phe;
R6 and R7 may be absent, or are independently Gly, Tyr, (D)- or (L)-Ala, or (D)- or (L)-Phe;
R8 is (D)- or (L)-Trp;
R9 is (D)- or (L)-Lys;
R10 is absent or is Gly, Abu, Cys, Thr, Val, (D)- or (L)-Ala, or (D)- or (L)-Phe;
R11 is Cys, (D)- or (L)-Ala, or (D)- or (L)-Phe;
R12 is absent or is Val, Thr, 1Nal or 2Nal; and
Y2 is amide, thioether, thioester or disulfide.
Preferably:
R4 is absent;
R5 is (D)- or (L)-Phe, or (D)- or (L)-Ala;
R6 may be absent and R6, when present, and R7 are independently (D)- or (L)-Phe, Ala or Tyr;
R10 is absent or is Thr, Val or (D)- or (L)-Phe;
R11 is (D)- or (L)-Ala, or (D)- or (L)-Phe; and
R12 is absent.
Alternatively:
R5 is (D)- or (L)-Ala, or (D)- or (L)-Phe;
R6 is absent or is (D)- or (L)-Ala, or (D)- or (L)-Phe;
R7 is (D)- or (L)-Ala, or (D)- or (L)-Phe;
R10 is absent or is Thr, Cys, (D)- or (L)- Ala;
R11 is Cys, (D)- or (L)-Ala, or (D)- or (L)-Phe; and
R12 is absent or is Thr.
Another embodiment has the general formula: 
wherein: m and n are 1 to 5
R6 is (D)- or (L)-Ala, or (D)- or (L)-Phe;
R7 is absent or is Tyr, (D)- or (L)- Ala, or (D)- or (L)-Phe;
R10 is Thr, Val, Cys or (D)- or (L)-Ala;
R11 is Cys, (D)- or (L)-Ala, or (D) or (L)-Phe;
R12 is Thr; and
Y2 is amide, thioether, thioester or disulfide.
Preferably:
R6 is (D)- or (L)-Ala;
R7 is absent or is (D)- or (L)-Phe;
R10 is Thr;
R11 is Cys; and
X is an alcohol group.
Yet another embodiment has the general formula: 
wherein:
the dotted line indicates that the bridge is connected to NR6 or NR7 at one end and to NR11 or NR12 at the other end;
R6 is absent or is (D)- or (L)-Phe or Ala;
R7 is (D)- or (L)-Phe, Ala or Tyr;
R8 is Thr, Ala, Val or Cys;
R11 is absent or is (D)- or (L)-Phe, Ala or Cys;
R12 is absent or is Thr or Thr reduced to an alcohol; and
Y2 is amide, thioether, thioester or disulfide.
Preferably, the bridge is connected to NR6 and NR11 or to NR6 and NR12 with R12 being Thr reduced to an alcohol.
Another preferred embodiment has the general formula: 
wherein m and n are 1 to 5;
X designates a terminal carboxy acid, amide or alcohol group;
R6 is (D)- or (L)-Phe or Tyr;
R7 is (D)- or (L)-Trp, (D)- or (L)-Phe, (D)- or (L)-1Nal or (D)- or (L)-2Nal, or Tyr;
R10 is Thr, Gly, Abu, Ser, Cys, Val, (D)- or (L)-Ala, or (D)- or (L)-Phe;
R11 is (D)- or (L)-Phe or (D)- or (L)-Ala;
R12 is Gly, Val, or (D)- or (L)-Phe; and
Y2 is thioether, thioester or disulfide.
Preferably:
R6 is Phe;
R7 is Trp;
R10 is Thr;
R11 is Phe;
R12 is Gly; and
Y2 is disulfide.
Another preferred embodiment has the general formula: 
wherein m and n are 1 to 5;
X designates a terminal carboxy acid, amide or alcohol group;
R4 is (D)- or (L)-Phe or Tyr;
R6 is (D)- or (L)-Phe or Tyr;
R7 is (D)- or (L)-Trp,(D)- or (L)-Phe, (D)- or (L)-1Nal or (D)- or (L)-2Nal, or Tyr;
R10 is Thr, Gly, Abu, Ser, Cys, Val, (D)- or (L)-Ala, or (D)- or (L)-Phe;
R11 is (D)- or (L)-Phe or (D)- or (L)-Ala;
R12 is Gly, Val, or (D)- or (L)-Phe; and
Y2 is thioether, thioester or disulfide.
Preferably:
R4 is (D)Phe;
R6 is Phe;
R7 is Trp;
R10 is Thr;
R11 is Phe;
R12 is Gly; and
Y2 is disulfide.
Another more preferred embodiment has the general formula: 
wherein m and n are 1 to 5;
X designates a terminal carboxy acid, amide or alcohol group;
R5 is (D)- or (L)-Phe or (D)- or (L)-Ala;
R7 is (D)- or (L)-Trp, (D)- or (L)-Phe, (D)- or (L)-1Nal or (D)- or (L)-2Nal, or Tyr;
R10 is Thr, Gly, Abu, Ser, Cys, Val, (D)- or (L)-Ala, or (D)- or (L)-Phe;
R12 is Gly, Val, or (D)- or (L)-Phe;
R13 is (D)- or (L)-Phe or (D)- or (L)-Ala; and
Y2 is amide, thioether, thioester or disulfide.
Preferably:
R5 is Phe;
R7 is Phe;
R10 is Thr;
R12 is Gly, Val, or (D)- or (L)-Phe;
R13 is Phe; and
Y2 is amide.
Additional preferred embodiments were synthesized using multiple peptide parallel synthesis (under the name TY-30005) comprise heptapeptide and octapeptide analogs in four groups (A-D) as described below.
Group A: 
wherein: m and n are 1 to 5;
X designates a terminal carboxy acid, amide or alcohol group;
R5 is absent or is 2Nal;
R6 is Phe(N2) or Gly(N3);
R7 is (p-Cl)Phe, (pxe2x80x94NH2)Phe, (p-F)Phe, (p-NO2)Phe or ChxGly;
R10 is Val, Gly, or (D)ChxGly;
R11 is Trp(C3) or GlyC2;
R12 is 2Nal or Thr;
Y2 is amide, thioether, thioester or disulfide.
Group B: 
wherein: m and n are 1 to 5;
X designates a terminal carboxy acid, amide or alcohol group;
R7 is (p-Cl)Phe, (p-NH2)Phe, (p-NO2)Phe, or Tyr;
R11 is Ile, Val or Ala;
Y2 is amide
Group C: 
wherein: m and n are 1 to 5;
X designates a terminal carboxy acid, amide or alcohol group;
R10 is Ala, Abu, Nle, Val or Thr;
R11 is Phe, Tyr, (p-Cl)Phe, (p-NH2)Phe, (p-NO2)Phe or (p-F)Phe;
Y2 is amide, thioether or thioester.
Group D: 
wherein: m and n are 1 to 5;
X designates a terminal carboxy acid, amide or alcohol group;
R6 is Val, Phe, (p-F)Phe or (p-Cl)Phe;
R7 is Trp, Tyr, (p-Cl)Phe, (pxe2x80x94NH2)Phe, (p-F)Phe, (p-NO2)Phe or ChxGly;
R11 is Val or ChxGly;
Y2 is amide.
The preferred analogs of the TY-30005 groups described in table 2 below:
The most preferred backbone cyclized somatostatin analogs of the invention described in table 3:
where X is xe2x80x94NH2 or xe2x80x94OH and the bridging group extends between the two building units or as indicated below:
For PTR 3171 and PTR 3173, the asterisk denotes that the bridging group is connected between the Nxcex1-xcfx89-functionalized derivative of an amino acid and the N terminus of the peptide. For PTR 3197 and PTR 3207, the asterisk denotes that the bridging group is connected between the Nxcex1-xcfx89-functionalized derivative of an amino acid and the side chain of the Cys residue. PTR 3205 is a bicyclic compound in which one bridge connects the two building units (Phe-C3 and Phe-N3) and the second is a disulfide bridge formed between the two Cys residues.
Somatostatin is a tetradecapeptide hormone whose numerous regulatory functions are mediated by a family of five receptors, whose expression is tissue dependent. Receptor specific analogs of somatostatin are believed to be valuable therapeutic agents in the treatment of various diseases. Attempts to design small peptide analogs having this selectivity have not been highly successful. It has now unexpectedly been found that the conformationally constrained backbone cyclized somatostatin analogs of the present invention, are highly selective to SST receptor subtypes.
The backbone cyclic peptides of this invention are novel selective analogs and preferably bind with higher affinity to a single receptor of the somatostatin receptor family. PTR 3113 and PTR 3123 are selective for the type 3 somatostatin receptor previously studied analogs have failed to achieve specificity to this receptor subtype. PTR 3183, 3185 and 3201 are selective for the type 5 somatostatin receptor. PTR 3209 is selective for the type 1 receptor. PTR 3203 is selective for receptors 3 and 5, and PTR 3173 is selective for receptors 2 and 5. PTR 3205 is a bicyclic analog which is selective to somatostatin receptor type 2.
The amino acid sequence of the corresponding backbone hexacyclic analogs (PTRs 3113, 3123 and 3171)is based on what are believed to be the most important amino acids derived from the native SRIF-14. From the data in the literature (SMS 210-995: A very potent and selective octapeptide analogue (i.e., Octreotide) of somatostatin having prolonged action, Bauer, et al. Life Sciences, 1982, 31, pp. 1133-1140), it was concluded that the amino acids of the native SRIF-14 in at least positions seven through 10, namely 7-Phe, 8-Trp, 9-Lys, and 10-Thr, and preferably positions six through 10, namely 6-Phe, 7-Phe, 8-Trp, 9-Lys, and 10-Thr, are essential to the pharmacophore of the hormone.
The present innovative backbone analogs preferably include 5 to 8 amino acids with special amine acid modifications. For certain preferred analogs, the amino acid Asn was substituted by the backbone Phe building unit at position 5. The configuration substitution of the native L-Trp at position 8 to D-Trp was made to improve the stability of the analog. The Thr residue at position 10 was deleted and the sequence completed by the corresponding backbone Phe building unit. The unique configuration substitution at position 9 from L-Lys to D-Lys as shown in PTRs 3123 and 3171 in comparison to PTR 3113 imparts improved selectivity of binding to the SST receptor subtype SSTR3 rather than SSTR5.
In certain most preferred analogs (PTR 3171 and 3173 for example) the bridge is connected between Nxcex1-xcfx89-functionalized derivative of an amino acid and the N-terminus of the peptide sequence.
The present novel analogs provide an additional dimension to the novelty of the backbone cyclization technology, in the utilization of a shortened backbone bridge (i.e., only one to three methylenes beside the peptide bond). This approach enables one to obtain much greater rigidity of the peptide, and to further constrain the desired conformation of the native pharmacophore.
An additional advantage of the hexapeptide analogs is related to their relative low molecular weight (their sequence consisting of only six amino acids), up to only 1000 daltons, in comparison to the most common somatostatin synthetic analogs which usually are hepta or octapeptides.
PTR 3123, 3173 and their analogs are as biostable as the conventional peptide Octreotide, and are much more stable than the native hormone SRIF-14, as tested in vitro against degradation of the most aggressive enzyme mixture in the body (e.g., renal homogenate) for up to 24 hours.
The peptide analogs induced physiological selectivity in vivo. PTRs 3113, 3123 and 3171 inhibit glucagon secretion but not growth hormone or insulin while PTR 3173 inhibit the secretion of both glucagon and growth hormone but not the secretion of insulin. The native hormone SRIF as well as its synthetic analog Octreotide, inhibit simultaneously growth hormone, glucagon and insulin and therefore they are not selective.
The special feature of the novel backbone cyclic peptide analogs PTR 3173 and 3123 is their physiological selectivity. PTR 3173 inhibits growth hormone and glucagon but not insulin, therefore it has the advantage of not causing hyperglycemia. In addition it is selective to malignancies expressing SSTR-2 and SSTR-5 only. PTR-3123 inhibits only the release of glucagon, which makes it a potential therapeutic agent for glucagonoma with no adverse effects on the release of growth hormone and insulin. In addition, it is an anticancer candidate for malignancies expressing SSTR-3 only.
PTR 3205 is a bicyclic compound in which one bridge connects the two building units and the second is a disulfide bridge formed between two Cys residues. This analog is selective for somatostatin receptor 2 and thus it is an anticancer candidate for malignancies expressing this receptor subtype without influencing other somatostatin receptor activities.
PTRs 3113, 3123, 3171 were found to be safe when administered intravenously to rats in a single dose of 6 mg/kg. PTR 3173 was found to be safe when administered intravenously to mice in a single dose of 3 mg/kg.
Resin:
1 g Rink amide or Tenta-gel resin, with loading of 0.2-0.7 mmol/g.
Fmoc-deprotection:
With 7 mL of 20% piperidine in NMP. Twice for 15 minutes following 5 washes with 10 mL NMP for 2 minutes with shaking.
Couplings:
1. Regular couplings (coupling to simple amino acids): with a solution containing 3 equivalents amino acid, 3 equivalents PyBroP and 6 equivalents of DIEA in 7 mL NMP. For 0.5-2 hours with shaking. Coupling is monitored by ninhydrine test and repeated until the ninhydrine solution become yellow.
2. Coupling of His and Asn with a solution containing 5 equivalents DIC and 5 equivalents HOBT in 10 mL DMF.
3. Coupling to Gly building units: with a solution containing 3 equivalents amino acid, 3 equivalents PyBroP and 6 equivalents DIEA in 7 mL NMP. Twice for 1-4 hours with shaking.
4. Coupling to building units which are not Gly: with a solution containing 5 equivalents amino acid, 1.5 equivalents triphosgene and 13 equivalents collidine in 15 mL dioxane or THF. Twice for 0.5-2 hours at 50xc2x0 C. with shaking.
Removal of the Allyl and Alloc protecting groups of the building units:
With 1.5 equivalents per peptide of Pd(PPh3)4 in 30 mL DCM containing 5% acetic acid and 2.5% NMM. For 1-4 hours with shaking.
Cyclization:
With a solution containing 3 equivalents PyBOP and 6 equivalents DIEA in 7 mL NMP. For 0.5-2 hours with shaking. Cyclization is monitored by ninhydrine test and repeated if necessary.
Cleavage:
With 82%-95% TFA supplemented with scavengers: 1-15% H2O, 1-5% TIS and 1-5% EDT.
Purification:
An individual purification method for each backbone cyclic peptide is developed on analytical HPLC to give the maximum isolation of the cyclic peptide from other crude components. The analytical method is usually performed using a C-18 Vydac column 250xc3x974.6 mm as the stationary phase and water/ACN containing 0.1% TFA mixture gradient.
The preparative method is designed by implying the analytical separation method on the 2xe2x80x3 C-18 Vydac preparative method. During the purification process, the peak containing the cyclic peptide is collected using a semi-automated fraction collector. The collected fractions are injected to the analytical HPLC for purity check. The pure fractions are combined and lyophilized.
The combined pure lyophilized material is analyzed for purity by HPLC, MS and capillary electrophoresis and by amino acid analysis for peptide content and amino acid ratio determination.
Preparation of Peptides with Backbone to Side Chain Cyclization
One preferred procedure for preparing the desired backbone cyclic peptides involves the stepwise synthesis of the linear peptides on a solid support and the backbone cyclization of the peptide either on the solid support or after removal from the support. The C-terminal amino acid is bound covalently to an insoluble polymeric support by a carboxylic acid ester or other linkages such as amides. An example of such support is a polystyrene-co-divinyl benzene resin. The polymeric supports used are those compatible with such chemistries as Fmoc and Boc and include for example PAM resin, HMP resin and chloromethylated resin. The resin bound amino acid is deprotected for example with TFA and to it is coupled the second amino acid, protected on the Nxcex1 for example by Fmoc, using a coupling reagent like BOP. The second amino acid is deprotected using for example piperidine 20% in DMF. The subsequent protected amino acids can then be coupled and deprotected at ambient temperature. After several cycles of coupling and deprotection that gives peptide, an amino acid having for example carboxy side chain is coupled to the desired peptide. One such amino acid is Fmoc-aspartic acid t-butyl ester. After deprotection of the Nxcex1 Fmoc protecting group, the peptide is again elongated by methods well known in the art. After deprotection a building unit for backbone cyclization is coupled to the peptide resin using for example the coupling reagent BOP. One such building unit is for example Fmoc-Nxcex1-(xcfx89-Boc-amino alkylene)amino acid. After deprotection the peptide can then be elongated, to the desired length using methods well known in the art. The coupling of the protected amino acid subsequent to the building unit is performed by such coupling agents exemplified by PyBrOP to ensure high yield. After the linear, resin bound peptide, has been prepared the co-alkylene-protecting groups, for example Boc and t-Bu, are removed by mild acid such as TFA.
The resin bound peptide is then divided into several parts. One part is subjected to on-resin cyclization using for example TBTU as cyclization agent in DMF to ensure high yield of cyclization, to give the N-backbone to side chain cyclic peptide resin. After cyclization on the resin the terminal amino protecting group is removed by agents such as piperidine and the backbone to side chain cyclic peptide is obtained after treatment with strong acid such as HF. Alternatively, prior to the removal of the backbone cyclic peptide from the resin, the terminal amino group is blocked by acylation with agents such as acetic anhydride, benzoic anhydride or any other acid such as adamantyl carboxylic acid activated by coupling agents such as BOP.
The other part of the peptide-resin undergoes protecting of the side chains used for cyclization, for example the xcfx89-amino and carboxy groups. This is done by reacting the xcfx89-amino group with for example Ac2O and DMAP in DMF and activating the free xcfx89-carboxy group by, for example, DIC and HOBT to give the active ester which is then reacted with, for example, CH3NH2 to give the non-cyclic analog of the cyclic peptide. Removal of the peptide from the resin and subsequent removal of the side chains protecting groups by strong acid such as HF to gives the non-cyclic analog of the backbone to side chain cyclic peptide.
The linear and/or non-cyclic analogs are used as reference compounds for the biological activity of their corresponding cyclic compounds.
General Screening of Somatostatin Analogs
The resulting somatostatin analogs are typically tested in vitro for their inhibition of the natural peptide (SRIF-14) binding to its 7-transmembranal receptors, and for their influence on second messengers and cell growth; and in vivo for inhibition of hormones and enzyme secretion.
The analogs can be further tested in vitro for their influence on cyclic adenosine monophosphate (cAMP) levels, tyrosine phosphatase activity, growth hormone secretion, and cell growth. The libraries can be further tested in vivo for the inhibition of growth-hormone release, and amylase, gastric acid, insulin and glucagon secretion in animals.
Conformationally constrained somatostatin analogs constructed based in part on the sequences of a number of known biologically active peptides or based on previously unknown novel sequences are presented in the examples below. The following examples are intended to illustrate how to make and use the compounds and methods of this invention and are in no way to be construed as a limitation.